Fig. 3: DRP1 is required for the mechanoregulation of YAP/TAZ, proliferation and cell fate.
a, MCF10A-RAS cells were cultured on fibronectin hydrogels for 24 h and treated with DRP1 inhibitors. The percentages of cells displaying endogenous YAP immunofluorescence staining prevalently in the cytoplasm (Cyto), evenly distributed between the cytoplasm and nucleus (N = C) or prevalently in the nucleus (Nuc) were quantified (n = 176 (stiff), 135 (soft), 129 (soft DRP1A) and 67 cells (soft MDIVI1) from four experiments). Scale bar, 20 μm. b, qPCR in MCF10A-RAS cells cultured on collagen I-coated hydrogels for 24 h and treated with DRP1 inhibitors. The mRNA levels are relative to GAPDH levels and normalized to the stiff controls (n = 6 from three experiments). c, YAP localization in D2.0R cells stably expressing control (shCO) or Drp1 shRNAs and treated with ROCK–MLCK inhibitors (YM) for 3 h (n = 162 (DMSO shCO), 99 (YM shCO), 142 (shCO YM DRP1A), 162 (YM shDrp1a) and 142 cells (YM shDrp1b) from three experiments). Scale bar, 10 μm. d, YAP localization in WT and Drp1 KO MEFs cultured on fibronectin hydrogels for 24 h (n = 124 (WT stiff), 119 (WT soft), 137 (KO stiff) and 122 cells (KO soft) from three experiments). Scale bar, 10 μm. e, YAP localization in 3T3L1 cells cultured on large or small micropatterned islands and treated with Drpitor1a for 24 h (n = 38 (large), 61 (small) and 55 cells (small DRP1A) from three experiments). Scale bar, 10 μm. f, YAP localization in MCF10A-RAS cells cultured under sparse (LD) and dense (HD) conditions and treated with Drpitor1a. Cells within five cell diameters from the scratch were quantified for comparison (n = 80 (LD), 452 (HD), 231 (HD DRP1A) and 225 cells (HD scratch) from three experiments). Scale bar, 10 μm. g, YAP localization in HUVECs conditioned by flow, with or without Drpitor1a (n = 1,098 (static), 1,032 (1.4 Pa), 1,098 (1.4 Pa DRP1A) and 3,421 cells (4.0 Pa) from two experiments). Scale bar, 30 μm. h, qPCR in HUVECs treated as in g. The mRNA levels are relative to GAPDH levels and normalized to the static controls (n = 6 from three experiments). i, EdU incorporation assay in MCF10A-RAS cells cultured on fibronectin hydrogels and treated with Drpitor1a for 24 h (n = 276 (stiff), 253 (soft) and 156 cells (soft DRP1A) from three experiments). j, EdU incorporation assay in MCF10A-RAS cells cultured as in f for 72 h. Drpitor1a was added for the last 24 h (n = 147 (LD), 1,003 (HD) and 1,109 cells (HD DRP1A) from three experiments). k, EdU incorporation assay in 3T3L1 cells cultured on large or small micropatterned islands and treated with Drpitor1a for 24 h (n = 60 (large), 51 (small) and 54 cells (small DRP1A) from three experiments). l,m, Dominant-negative HA-DRP1 (DRP1 DN) and constitutively active FLAG-TAZ-4SA (TAZ4SA) induce cholangiocellular markers (l) and EdU incorporation (m) in mouse liver (n = 4 mice). Scale bars, 5 μm. The data are presented as means ± s.d. (a and c–g) or means and single points (b and h–m). Statistical significance was determined by two-tailed Student’s t-test. In a and c–f, P values were calculated on nuclear YAP levels. Source numerical data are available.
